The major objective of this project is to uncover the molecular mechanisms of a variety of genetic rearrangements. The transposition reaction of bacteriophage Mu is studied as a model system. Critical steps in Mu transposition are a pair of DNA cleavages and strand transfers which generate a branched DNA intermediate. Efficient formation of this intermediate requires, besides the MuA protein which is the transposase, the following accessory factors: the E. coli-encoded HU and IHF proteins, ATP, and Mg++. MuA interacts with two different specific DNA sequences, one at the ends of the Mu genome and the other at the Mu operator. Interactions involving multiple MuA molecules, accessory protein factors and sequence elements on the donor DNA lead to formation of a stable protein-DNA complex in which the two Mu ends are synapsed by a tetramer of MuA. Next, a pair of single strand cuts are made to expose the 3' ends of the Mu DNA. The cleaved donor DNA remains tightly associated with the MuA tetramer and this complex efficiently captures a "target" DNA molecule provided it is provided it is bound by MuB. A staggered cut is introduced into the target DNA and the two 5' ends are joined to the 3' ends of the Mu DNA in a concerted reaction. The assembly process and the functional organization of the MuA tetramer-Mu DNA complex have been studied by making use of a variety of mutant MuA proteins with missing functional domains. Structurally and functionally important protein-DNA interactions within the stable complexes were analyzed by assembling the complexes from short Mu end DNA fragments and MuA under permissive reaction conditions, bypassing the need for many of the cofactors normally required for the process. The structure of the catalytic core domain of MuA has been determined by X- ray crystallography. The Mua catalytic subdomain shares remarkable similar structural arrangements with the core domain of HIV integrase which carried out similar biochemical reactions. In collaboration with scientists in LCP/NIDDK, the N-terminal domain of MuA was shown to have th "winged helix-turn-helix" type of DNA binding structure by NMR techniques.